Characterisation of weak and null phenotypes in the KEL and JK blood group systems

The focus of this thesis is on KEL and JK, two clinically important blood group systems, and the investigations of blood samples with aberrant antigen expression as in null and weak phenotypes, or with a phenotype that does not correspond to genotype. With the increasing use of DNA assays based on single nucleotide polymorphisms for blood group prediction, it is important to characterize the blood-groupencoding genes. Otherwise, when using these assays for phenotype prediction, tests risk giving rise to false results if null or otherwise altered genes are present and the test are not designed to take alterations into consideration. This is especially applicable for foetal blood group prediction, finding matching blood donors to transfusion-dependent patients or to correctly type multi-transfused patients where serology is inadequate. For each blood group system investigated here, four novel null alleles and one allele giving rise to weak antigen expression were characterised. Most importantly, a novel Jk(a+w) phenotype was described and associated with a novel JK*01M allele. A control cohort was screened for the associated polymorphism and allele frequencies calculated. A simple PCR-ASP method for screening of five null alleles giving rise to the Jk(a–b–) phenotype was devised, utilised and proven useful. In summary, this thesis contributes to the overall understanding of the molecular genetic basis of human blood group diversity in the two investigated systems and the results will improve the prediction of blood group phenotypes from DNA-based assays

A simple screening assay for the most common JK*0 alleles revealed compound heterozygosity in Jk(a-b-) probands from Guam

The Jk(a-b-) phenotype results from alterations in the JK gene and is characterized by absence of the RBC urea transporter in the cell membrane. The frequency of Jk(a-b-) varies among populations, but this phenotype is most commonly found in people of Polynesian and Finnish descent. Although rare, Jk(a-b-) individuals present a clinical challenge because anti-Jk3 is produced readily in response to transfusion and pregnancy, and Jk(a-b-) blood is not routinely available. Identification of Jk(a-b-) patients and donors is most often performed serologically. However, ten JK*o alleles have been identified, and this information can be used in DNA-based typing. We selected five JK*o alleles that had been encountered by our reference laboratory in two or more samples from unrelated individuals and designed an allele-specific primer PCR assay for use as an initial screening tool. After in-house validation, we tested genomic DNA from a family: a mother and her two sons referred to us for genetic investigation of their Jk(a-b-) phenotypes. Two different nucleotide substitutions, -1g>a in intron 5 (IVS5) and 956C>T in exon 10, originally associated with Polynesian and Indian/African populations respectively, were identified in the family. The mother and one son were compound heterozygotes, and the second son was homozygous for IVS5-1g>a. We conclude that the effort to design and validate such a screening assay was cost-efficient when compared with DNA sequencing costs. Furthermore, selection of the more common JK*o mutations was a practical approach that resulted in rapid identification of the genetic bases behind the Jk(a-b-) phenotypes in this unusual family. Although an obvious target for eventual inclusion into high-throughput genotyping platforms for clinical diagnostic services, current systems are very limited. Our approach provides a simple and inexpensive method for the identification of these rare alleles